Quantification of Exosomal Markers in Serum

Serum levels of EVs expressing either platelet markers (CD41, CD61) or EV marker (CD63) were quantified using the PS Capture Exosome ELISA Kit (Streptavidin HRP) (Wako Pure Chemical Industries, Ltd) as described previously [9 (link)]. Briefly, 100-fold diluted sera (100 μl) were incubated with Tim4-coated plates for 2 h at room temperature (RT). After washing, a biotinylated antibody for each marker protein was incubated for 1 h at RT. Then, horseradish peroxidase (HRP)-labeled streptavidin was incubated for 2 h at RT followed by washing. After incubation with 3,3’,5,5’-tetramethylbenzidine solutions for 30 min, a stop solution was applied. An optical density (OD) was measured at 450 nm as the dominant wavelength and 620 nm as the secondary wavelength. The following antibodies were biotinylated and used for sandwich assays: anti-CD41 mAb (1 μg/mL, clone No. 745201; R&D Systems), anti-CD61 mAb (1 μg/mL, clone No. 256809; R&D Systems), anti-CD63 (component of PS Capture Exosome ELISA Kit; Wako).
CA19-9 was measured using Accuraseed CA19-9 reagents (FUJIFILM Wako Pure Chemical Co., Osaka, Japan).